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Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
773
Ion Channels Are Gated by Ligands and by
Membrane Potential
In a fourth class of signal transducers, receptors are coupled directly or
indirectly to ion channels in the plasma membrane. The best-understood example of such a receptor is the nicotinic acetylcholine receptor, which responds to the neurotransmitter acetylcholine. It is
found in the postsynaptic cells in certain nerve synapses (Fig. 22-34)
and in the junction between a muscle fiber and the neuron that controls it. The acetylcholine receptor complex (Mr 250,000) is composed of
four different polypeptide chains, one of which is present in two copies.
The transmembrane arrangement of these five chains provides a hydrophilic channel through which ions can traverse the lipid bilayer.
When acetylcholine released from the presynaptic nerve ending binds
Voltage- +
gated Na+
channel (site
of action of
tetrodotoxin
and
saxitoxin)
Axon of
presynaptic
neuron
Action
potential
Voltage-' 2+ +S
gated Ca A
channel
Secretory
vesicles containing
acetylcholine
Synaptic
cleft
Cell body of
postsynaptic
neuron
Acetylcholine receptor-ion
channels (site of action of
tubocurarine, cobrotoxin,
bungarotoxin)
/
Figure 22-34 Role of voltage-gated and ligandgated ion channels in passage of an electrical signal
between two neurons. Initially, the plasma membrane of the presynaptic neuron is polarized, with
the inside negative; this results from the action of
the electrogenic Na+K+ ATPase, which pumps three
Na+ outward for every two K+ pumped into the
neuron (see Fig. 10-22). (T) A stimulus to this neuron causes an action potential to move downward
along its axon (white arrow). The opening of one
voltage-gated Na+ channel allows NaT entry, and
the resulting local depolarization causes the adjacent Na+ channel to open, and so on. The directionality of movement of the action potential is ensured
by the brief refractory period that follows the opening of each voltage-gated Na+ channel. (2) When
this wave of depolarization reaches the axon tip,
voltage-gated Ca2+ channels open, allowing Ca2^
entry into the presynaptic neuron. (3) The resulting
increase in internal [Ca2+] triggers exocytosis of the
neurotransmitter acetylcholine into the space between the neurons (synaptic cleft). (4) Acetylcholine
binds to its specific receptor in the plasma membrane of the cell body of the postsynaptic neuron,
causing the ligand-gated ion channel that is part of
the receptor to open. (5) Extracellular Na^ and K+
enter through this channel, depolarizing the postsynaptic cell. The electrical signal has thus passed
to the postsynaptic cell, and will move along its
axon to a third neuron by this same sequence of
events. The effects of the toxins shown in parentheses are discussed on p. 774.
CH 3 -C
P
X
Action
potential
/
I
O—CH 2 -CH 2 -N(CH 3 ) 3
Acetylcholine
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Part III Bioenergetics and Metabolism
to its receptor in the postsynaptic cell (Fig. 22-34), the receptor-ion
channel opens, allowing transmembrane passage of Na+ and K+ ions
(pp. 292-293). The receptor is therefore referred to as a ligand-gated
ion channel. The resulting depolarization of the postsynaptic membrane triggers muscle contraction or initiates an action potential in the
postsynaptic neuron.
The action potential is a wave of transient depolarization that
sweeps the neuron from the site of the initial stimulus (in the cell body
of the neuron), along the long, thin cytoplasmic extension (axon), to the
next synapse. Essential to this signaling mechanism are several types
of "voltage-gated" ion channels in the plasma membrane of the neuron.
These channels, formed by transmembrane proteins, open and close in
response to changes in the transmembrane electrical potential. Along
the entire length of the axon are voltage-gated Na+ channels (Fig.
22-34), which are closed when the membrane is polarized, but open
briefly when the membrane potential is reduced (i.e., during depolarization). After each opening of a Na+ channel there follows a brief
refractory period during which the channel cannot open again, and
thus a unidirectional wave of depolarization sweeps from the nerve cell
body toward the end of the axon.
At the distal tip of the neuron are voltage-gated Ca2+ channels.
When the wave of depolarization reaches these channels they open,
letting Ca2+ enter from the extracellular space and triggering acetylcholine release into the synaptic cleft (Fig. 22-34). Acetylcholine diffuses to the postsynaptic cell, where it binds to acetylcholine receptors;
thus the message is passed to the next cell in the circuit.
Toxins, Oncogenes, and Tumor Promoters
Interfere with Signal Transductions
Biochemical studies of signal transductions have led to an improved
understanding of the pathological effects of toxins produced by the bacteria that cause cholera and pertussis (whooping cough). Both toxins
are enzymes that interfere with normal signal transductions in the
host animal. Cholera toxin, secreted by Vibrio cholerae found in contaminated drinking water, catalyzes the transfer of ADP-ribose from
NAD+ to the a subunit of Gs, blocking its GTPase activity (Fig. 22-26)
and thereby rendering it permanently activated (Fig. 22-35). This results in continuous activation of the adenylate cyclase of intestinal
epithelial cells, and the resultant high concentration of cAMP triggers
continual secretion of Cl~, HCO3 , and water into the intestinal lumen.
The resulting dehydration and electrolyte loss are the major pathologies in cholera. The pertussis toxin produced by Bordetella pertussis
catalyzes ADP-ribosylation of Gi? preventing GDP displacement by
GTP and blocking inhibition of adenylate cyclase by Gi; this defect
produces the symptoms of whooping cough, including hypersensitivity
to histamines and lowered blood glucose.
The critical importance of ligand- and voltage-gated ion channels
in nerve signal conduction as described above is clear from the effects
of several naturally occurring toxins. Tubocurarine, the active component of curare (used as an arrow poison in the Amazon), and toxins
from snake venoms (cobrotoxin and bungarotoxin), block the acetylcholine receptor or prevent the opening of its ion channel (Fig. 2234). By blocking signals from nerves to muscles, these toxins cause
paralysis and death. Tetrodotoxin (from the internal organs of puffer
fish) and saxitoxin (produced by the marine dinoflagellate that occa-
Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
775
0
II
i
•Arg-NH2
+
i
°
°
II
II
- _
CH 2 -O-P—0—P-0- Rib — Adenine
,
|
|
k y ^
^N^O
H\L__J/H
Normal Gs: GTPase activity
terminates the signal
from receptor to adenylate
cyclase.
HO
OH
NAD+
O
I
cholera
toxin !
C—NH 2
O
CH9—O—P—O—P—O — Rib — Adenine
I
O"
O"
Arg-NH/°
ADP-ribosylated Gs:
GTPase activity is inactivated;
Gs constantly activates
adenylate cyclase.
O
OH
sionally causes "red tides") are also deadly poisons, which block neurotransmission by preventing the opening of Na + channels.
Tumors and cancer are the result of uncontrolled cell division. Normally, cell division is highly regulated by a family of growth factors,
proteins that cause resting cells to undergo cell division and, in some
cases, differentiation. Some growth factors are cell type-specific, stimulating division of only those cells with appropriate receptors; other
growth factors are more general in their effects. Among the well-studied growth factors are epidermal growth factor (EGF), nerve growth
factor (NGF), fibroblast growth factor (FGF), platelet-derived growth
factor (PDGF), erythropoietin, and a family of proteins called lymphokines, which includes interleukins (IL-1, IL-2, etc.) and interferon y.
There are also extracellular factors that antagonize the effects of
growth factors, slowing or preventing cell division; transforming
growth factor )8 (TGF/3) and tumor necrosis factor (TNF) are such factors.
These extracellular signals act through cell-surface receptors very
similar to those for hormones, and by similar mechanisms: the production of intracellular second messengers, protein phosphorylation, and
ultimately, alteration of gene expression.
It is becoming clear that many types of cancer are the result of
abnormal signal-transducing proteins, which lead to continual production of the signal for cell division. The mutated genes that encode these
defective signaling proteins are oncogenes. (Oncogenes, and gene
function in general, are discussed in Chapter 25.) Oncogenes were originally discovered in tumor-causing viruses, then later found to be
closely similar to or derived from genes present in the animal host
cells. Most likely, these viral genes originated from normal host genes
(proto-oncogenes) that encode growth-regulating proteins. During certain types of viral infections, these DNA sequences can be copied by the
virus and incorporated into its genome (Fig. 22-36). At some point
during the cycle of viral infection, the gene can become defective as a
ADP-ribose
Figure 22-35 The toxins produced by the bacteria
that cause cholera and whooping cough (pertussis)
are enzymes that catalyze transfer of the ADPribose moiety of NAD^ to an Arg residue of G proteins: Gs in the case of cholera (as shown here) and
Gj in whooping cough. The G proteins thus modified fail to respond to normal hormonal stimuli.
The pathology of both diseases results from defective regulation of adenylate cyclase and overproduction of cAMP.
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Part III Bioenergetics and Metabolism
Retrovirus
Normal cell
is infected with
retrovirus.
Gene for regulatory
growth protein
(proto-oncogene)
Host cell now has
retroviral genome
incorporated near
proto-oncogene.
Forming virus
encapsulates
proto-oncogene
and viral genome.
Retrovirus with
proto-oncogene
Infection cycles
Mutation creates
oncogene.
Retrovirus with
oncogene invades
normal cell.
Transformed cell,
producing defective
regulatory protein
Figure 22-36 Conversion of a normal regulatory
gene into a viral oncogene. (T) A normal cell is infected by a retrovirus, which (2) inserts its own
genome into the chromosome of the host cell, near
the gene for a regulatory protein (the proto-oncogene). (3) Virus particles released from the infected
cell infrequently "capture" a host gene, in this case
the proto-oncogene that encodes a regulatory protein. (4) During several cycles of infection, a mutation occurs in the viral proto-oncogene, converting
it into an oncogene. (5) When the virus subsequently infects a normal cell, it introduces the oncogene into the host-cell DNA. Transcription of the
oncogene leads to the production of a defective regulatory protein that continuously gives the signal
for host-cell division, overriding normal mechanisms for limiting cell division. Host cells infected
with oncogene-carrying viruses therefore undergo
unregulated cell division—they form tumors.
Proto-oncogenes can also undergo mutation to oncogenes without the intervention of a retrovirus;
these cellular oncogenes also confer unregulated
growth on the cells in which they occur.
result of truncation or some other mutation. During a subsequent infection, when this viral oncogene is expressed in its host cell, the abnormal protein product interferes with normal regulation of cell
growth, and the unregulated growth can result in a tumor. Oncogenes
can also arise from proto-oncogenes without viral involvement. Chromosomal rearrangements, chemical agents, radiation, or other factors
can cause mutations in the genes that encode signal-transducing proteins. The resulting oncogenes express defective proteins and defective
signaling, once again leading to tumor growth.
Many viral oncogenes encode unregulated tyrosine kinase activities, and in some cases the oncogene product is nearly identical to a
normal animal-cell receptor, but with the normal signal-binding site
defective or missing. For example, the erbB oncogene product, a protein called ErbB, is essentially identical to the normal receptor for
epidermal growth factor, except that ErbB lacks the domain that normally binds EGF (Fig. 22-37, p. 777). The erbB2 oncogene is commonly
associated with adenocarcinomas (cancers) of the breast, stomach, and
ovary.
Other signal-transducing proteins with oncogene analogs are the
GTP-binding (G) proteins. One well-characterized oncogene, ras, encodes a protein with normal GTP binding but no GTPase activity.
When the Ras protein (p. 682) is produced in an animal cell, it remains
always in the activated form, regardless of the signals coming through
normal receptors. Again, the result is unregulated growth—cancer.
Mutations in ras are associated with 30 to 50% of lung and colon carcinomas and over 90% of pancreatic carcinomas.
The action of a group of compounds known as tumor promoters
can also be understood in the light of what we know of signal transduction. The best understood of these compounds, phorbol esters, are
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Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
chemically synthesized compounds that are potent activators of protein kinase C. They apparently mimic cellular diacylglycerol as second
messengers (Fig. 22-32), but unlike naturally occurring diacylglycerols they are not rapidly metabolized. By permanently activating protein kinase C, these synthetic tumor promoters interfere with the normal regulation of cell growth and division.
Protein Phosphorylation and Dephosphorylation
Are Central to Cellular Control
One common denominator in signal transductions—whether they involve adenylate cyclase, a transmembrane receptor-tyrosine kinase,
phospholipase C, or an ion channel—is the eventual regulation of the
activity of a protein kinase. We have seen examples of kinases activated by cAMP, insulin, Ca2+/calmodulin, Ca2+/diacylglycerol, and by
phosphorylation catalyzed by another protein kinase. The number of
known protein kinases has grown remarkably since their discovery by
Edwin G. Krebs and Edmond H. Fischer in 1959. Hundreds of different
protein kinases, each with its own specific activator and its own specific protein target(s), may be present in eukaryotic cells. Although
many other types of covalent modifications are known to occur on proteins, it is clear that phosphorylations make up the vast majority of
known regulatory modifications of proteins.
The addition of a phosphate group to a Ser, Thr, or Tyr residue
introduces a bulky, highly charged group into a region that was only
moderately polar. When the modified side chain is located in a region
of the protein critical to its three-dimensional structure, phosphorylation can be expected to have dramatic effects on protein conformation
and thus on the catalytic activity of the protein. As a result of evolution, the kinase-phosphorylated Ser, Thr, and/or Tyr residues of regulated proteins occur within common structural motifs (consensus sequences) that are recognized by their specific protein kinases (Table
22-9).
Table 22-9 Consensus sequences for protein kinases
Protein kinase
Protein kinase A
Consensus sequence*
-X-R-(R/K)-X-(S/T)-X-
Protein kinase G
-X-(R/K) 2 _ 3 -X-(S/T)-X-
Protein kinase C
-X-tR/KLs, Xo_2)-(S/T)-(Xo_2,
Ca2+/calmodulin kinase II
Phosphorylase b kinase
Insulin receptor kinase
EGF receptor kinase
-X-R-X-X-(S/T)-X-K-R-K-Q-I-(S/T)-V-R-T-R-D-I-Y-E-T-D-Y-Y-R-K-T-A-E-N-A-E-Y-L-R-V-A-P
Source: Data from Kemp, B.E. & Pearson, R.B. (1990) Protein kinase recognition sequence motifs. Trends Biochem. Sci. 15, 342-346; and Kennelly, P.J. & Krebs, E.G. (1991) Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases.
J. Biol. Chem. 266, 15555-15558.
* (S/T) and Y are the Ser (or Thr) and Tyr residues that are phosphorylated. X is a less essential
residue; any of several amino acids may be at this position. Essential residues are indicated by
their one-letter abbreviations (see Table 5-1). The notation -(WKi_3, XQ_2)- means that at this
position there are from one to three amino acids, which can be R (Arg) or K (Lys), as well as
zero to two of any amino acids, in any sequence (the comma indicates that no sequence is implied).
CHoOH
Myristoylphorbol acetate
(a phorbol ester)
Extracellular space
EGF-binding
domain
Tyrosine kinase
domain
EGF binding site Binding of EGF
empty; tyrosine
activates
kinase is inactive, tyrosine kinase.
Normal EGF receptor
Tyrosine
kinase is
constantly
active.
ErbB protein
Figure 22-37 The product of the erbB oncogene
(the ErbB protein) is a truncated version of the
normal receptor for epidermal growth factor (EGF).
Its intracellular domain has the structure normally
induced by EGF binding, but the protein lacks the
extracellular binding site for EGF. Unregulated by
EGF, ErbB continuously signals cell division.
Part III Bioenergetics and Metabolism
Figure 22—38 The enzyme glycogen synthase contains at least nine separate sites in five designated
regions susceptible to phosphorylation by one of the
cellular protein kinases. The activity of this enzyme
is therefore capable of modulation in response to a
variety of second messengers produced in response
to different extracellular signals. Thus regulation is
a matter not of binary (on/off) switching but of
finely tuned modulation of the activity over a wide
range.
Glycogen
synthase
molecule
A B
ABC
45
1
I I
A B
COO"
Kinase
cAMP-dependent
protein kinase
cGMP-dependent
protein kinase
Phosphorylase b kinase
Ca2+/calmodulin-dependent
kinase
Glycogen synthase
kinase 3
Glycogen synthase
kinase 4
Casein kinase II
Casein kinase I
Protein kinase C
Glycogen synthase
sites phosphorylated
Degree of
synthase
inactivation
1A, IB, 2 , 4
1A, IB, 2
2
+
IB, 2
+
3A, 3B, 3C
+ ++
2
+
5
0
At least 9 sites
1A
+ + 4- +
+
Not all cases of regulation by phosphorylation are as simple as
those we have described. Some proteins have consensus sequences recognized by several different protein kinases, each of which can phosphorylate the protein and alter its enzymatic activity. For example,
glycogen synthase is inactivated by cAMP-dependent phosphorylation
of specific Ser residues, and is also modulated by at least four other
protein kinases that phosphorylate four other sites in the protein (Fig.
22-38). Some of the phosphorylations inhibit the enzyme more than
others, and some combinations of phosphorylation are cumulative. The
result of all of these regulations is the potential for extremely subtle
modulation of the activity of glycogen synthase, allowing very finely
tuned responses to varying metabolic circumstances.
The end effect of epinephrine's interaction with the /3-adrenergic
receptor is the phosphorylation of several cellular enzymes, including
glycogen synthase and glycogen phosphorylase. To serve as an effective
regulatory mechanism, this phosphorylation must be reversible, allowing the regulated enzymes to return to their prestimulus level when
the hormonal signal stops. In muscle, for example, the enzyme phosphoprotein phosphatase-1 dephosphorylates glycogen phosphorylase,
phosphorylase b kinase, and glycogen synthase (see Figs. 14-17, 1915), reversing the effects of cAMP on the activities of these enzymes.
This enzyme (sometimes called phosphorylase a phosphatase, synthase phosphatase, or kinase phosphatase to indicate its substrate
specificity) is regulated by another protein, phosphoprotein phosphatase inhibitor. This inhibitor, when phosphorylated by protein
kinase A, inhibits phosphoprotein phosphatase-1. A rise in the concentration of cAMP therefore stimulates phosphorylation of certain regulated proteins such as glycogen phosphorylase and also slows dephosphorylation of these proteins, prolonging the effect of phosphorylation.
Cells contain a family of phosphoprotein phosphatases that hydrolyze specific phosphoserine, phosphothreonine, and phosphotyrosine
Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
779
esters, releasing Pi. Although this class of enzymes is not yet as thoroughly studied as the protein kinases, it is very likely that these phosphatases will turn out to be just as important as the protein kinases in
regulating cellular processes and metabolism. The known phosphoprotein phosphatases show substrate specificity, acting on only a subset of
phosphoproteins, and they are in some cases regulated by a second
messenger or an extracellular signal. Some protein phosphatases are
transmembrane proteins of the plasma membrane, with extracellular
receptorlike domains and intracellular phosphatase domains; they
may well prove to be regulated by extracellular signals in a fashion
similar to regulation of the tyrosine kinase of the insulin receptor. The
complexity and the subtlety of the regulatory mechanisms achieved by
evolution strain the imagination, and the experimental challenges of
discovering the full range of regulatory mechanisms remain to be met.
Steroid and Thyroid Hormones Act in the
Nucleus to Change Gene Expression
The mechanism by which steroid and thyroid hormones exert their
effects is fundamentally different from that for the other types of hormones. Steroid hormones (estrogen, progesterone, and cortisol, for example), too hydrophobic to dissolve readily in the blood, are carried on
specific carrier proteins from the point of their release to their target
tissues. In the target tissue, these hormones pass through the plasma
membrane by simple diffusion and bind to specific receptor proteins in
the nucleus (Fig. 22-39). The hormone-receptor complexes act by
Serum binding protein
with bound hormone
I...
HRE
v
— ' Structural
gene
Translation
on ribosomes
Figure 22-39 The general mechanism by which
steroid and thyroid hormones, retinoids, and vitamin D act to regulate gene expression. (I) Hormone (H) carried to the target tissue on serum
binding proteins diffuses across the plasma membrane and binds to its specific receptor protein
(Rec) in the nucleus. (2) Hormone binding changes
the conformation of the receptor, allowing it to form
dimers in the nucleus with other hormone—receptor
complexes of the same type and to bind to specific
regulatory regions, hormone response elements
(HREs), in the DNA adjacent to specific genes.
(3) This binding somehow facilitates transcription
of the adjacent gene(s) by RNA polymerase (Chapter 25), increasing the rate of messenger RNA formation and (4) bringing about new synthesis of the
hormone-regulated gene product. The changed level
of the newly synthesized protein produces the cellular response to the hormone. The details of protein
synthesis are discussed in Chapter 26.
780
Part III Bioenergetics and Metabolism
binding to highly specific DNA sequences called hormone response
elements (HREs) (Fig. 22-39) and altering gene expression. Hormone
binding triggers changes in the conformation of the receptor proteins
so that they become capable of interacting with specific transcription
factors (Chapter 27). The bound hormone-receptor complex can either
enhance or suppress the expression (transcription into messenger
RNA; Chapter 25) of specific genes adjacent to HREs, and thus the
synthesis of the genes' protein products (Chapter 26).
The DNA sequences (HREs) to which hormone-receptor complexes
bind are similar in length and arrangement, but different in sequence,
for the various steroid hormones. The HRE sequences recognized by a
given receptor are very similar but not identical; for each receptor
there is a "consensus sequence" (Table 22-10), which the hormonereceptor complex binds at least as well as it binds the natural HREs.
Each HRE consensus sequence consists of two six-nucleotide sequences, either contiguous or separated by three nucleotides. The two
hexameric sequences occur either in tandem or in a palindromic arrangement (Fig. 12-20). The hormone-receptor complex binds to the
DNA as a dimer, with each monomer recognizing one of the six-nucleotide sequences. The ability of a given hormone to alter the expression
of a specific gene depends upon the HRE element's exact sequence and
on its position relative to the gene and the number of HREs associated
with the gene.
Table 22-10 Consensus sequences of some hormone response
elements
Hormone
Sequence of DNA (both strands)*
Glucocorticoid
(5') AGAACAXXXTGTTCT
(3') TCTTGTXXXACAAGA
(5') AGGTCAXXXTGACCT
(3') TCCAGTXXXACTGGA
(5') AGGTCATGACCT (3')
(3') TCCAGTACTGGA (5')
Estrogen
Thyroid
(3')
(5')
(3')
(5')
(strand 1)
(strand 2)
(strand 1)
(strand 2)
(strand 1)
(strand 2)
Source: Data from Schwabe, J.W.R. & Rhodes, D. (1991) Beyond zinc fingers: steroid hormone
receptors have a novel structural motif for DNA recognition. Trends Biochem. Sci. 16, 291-296;
and Fuller, P.J. (1991) The steroid receptor superfamily: mechanisms of diversity. FASEB J. 5,
3092-3099.
* X represents any nucleotide.
Comparison of the amino acid sequences of receptors for several
steroid hormones as well as receptors for thyroid hormone, vitamin D,
and retinoids has revealed several highly conserved sequences and
some regions in which the sequences differ considerably with receptor
type (Fig. 22-40). (Retinoids are compounds related to retinoate, the
carboxylate form of vitamin Ax (see Fig. 9-18), which have hormonelike
actions on some cell types.) A centrally located sequence of 66 to 68
residues is very similar in all of the receptors; this is the DNA-binding
region, which resembles regions of other proteins known to bind DNA.
All of these DNA-binding regions share the "zinc finger" structure (see
Fig. 27-12), a sequence containing eight Cys residues that provide
binding sites for two Zn 2+ ions, which stabilize the DNA-binding domain.
Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
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DNA binding
activation
(66-68 residues,
(variable sequence
highly
and length)
conserved)
Hormone binding
(variable sequence
and length)
The region of the hormone receptor responsible for hormone binding (the ligand-binding region, always at the carboxyl terminus) is
quite different in different members of the hormone receptor family.
The glucocorticoid receptor is only 30% homologous with the estrogen
receptor and 17% homologous with the thyroid hormone receptor. In
the vitamin D receptor, the ligand-binding region consists of only 25
residues, whereas it has 603 residues in the mineralocorticoid receptor. The different sequences are reflected in different specificities for
hormone binding. Mutations that change one amino acid residue in
this region result in loss of responsiveness to a specific hormone; some
humans unable to respond to cortisol, testosterone, vitamin D, or thyroxine have been shown to have such mutations in the corresponding
hormone receptor.
The specificity of the ligand-binding site is exploited in the use of a
drug, tamoxifen, in the treatment of breast cancer in humans. In
some types of breast cancer, division of the cancerous cells depends on
the continued presence of the hormone estrogen. Tamoxifen competes
with estrogen in binding to the estrogen receptor, but the tamoxifenreceptor complex is inactive in gene regulation. Consequently,
tamoxifen administration after surgery or chemotherapy for this type
of breast cancer slows or stops the growth of remaining cancerous cells,
prolonging the life of the patient.
Another steroid analog, the drug RU486, is used in the very early
termination of pregnancy. An antagonist of the hormone progesterone,
RU486 binds to the progesterone receptor and blocks hormone actions
essential to the implantation of the fertilized ovum in the uterus. As of
1992, RU486 had not been approved for use in the United States.
The ability of a given steroid or thyroid hormone to act on a specific
cell type depends not only on whether the receptor for that hormone is
synthesized by the cell, but also on whether the cell contains enzymes
that metabolize the hormone. Some hormones (testosterone, thyroxine,
vitamin D) are enzymatically converted into more active derivatives
within the target cell; others, such as cortisol, are converted to an inactive form in some cells, making these cells resistant to that hormone.
Figure 22-40 The DNA-binding domain common
to a number of steroid hormone receptor proteins.
These proteins have a binding site for the hormone,
a DNA-binding domain, and a region that activates
the transcription of the regulated gene. The DNAbinding region is highly conserved. The sequence
shown here (see Table 5-1 for amino acid abbreviations) is that for the estrogen receptor, but the residues in bold type are common to all such receptors.
Eight critical Cys residues bind to two Zn2+ ions
that stabilize the "zinc finger" structure shared
with many other DNA-binding proteins (see Fig.
27—12). The regulation of gene expression is described in more detail in Chapter 27.
CH3
Tamoxifen
CH
c=C—CH 3
RU486
(mifepristone)
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Part III Bioenergetics and Metabolism
In addition to the DNA-binding and ligand-binding regions, steroid
receptors also have two domains that interact (in a way not fully understood) with elements of the transcriptional (RNA-synthesizing)
machinery in the nucleus. The combination of DNA binding and this
interaction with the transcriptional apparatus allows the steroid hormone-receptor complex to modulate the rate at which proteins are
produced from a specific gene. The relatively slow action of steroid
hormones (hours or days are required for their full effect) is a consequence of their mode of action; time is required for RNA synthesis in
the nucleus and for the subsequent protein synthesis.
Summary
In mammals there is a division of metabolic labor
among specialized tissues and organs. Coordination of the body's diverse metabolic activities is
accomplished by hormonal signals that circulate in
the blood. The liver is the central distributing and
processing organ for nutrients. Sugars and amino
acids produced in digestion cross the intestinal epithelium and enter the blood, which carries them to
the liver. Some triacylglycerols derived from ingested lipids also make their way to the liver,
where the constituent fatty acids are used in a variety of processes. Glucose-6-phosphate is the key
intermediate in carbohydrate metabolism. It may
be polymerized into glycogen, dephosphorylated to
blood glucose, or converted to fatty acids via acetylCoA. It may undergo degradation by glycolysis and
the citric acid cycle to yield ATP energy or by the
pentose phosphate pathway to yield pentoses and
NADPH. Amino acids are used to synthesize liver
and plasma proteins, or their carbon skeletons
may be converted into glucose and glycogen by gluconeogenesis; the ammonia formed by their deamination is converted into urea. Fatty acids may be
converted by the liver into other triacylglycerols,
cholesterol, or plasma lipoproteins for transport to
and storage in adipose tissue. They may also be
oxidized to yield ATP, and to form ketone bodies to
be circulated to other tissues.
Skeletal muscle is specialized to produce ATP
for mechanical work. During strenuous muscular
activity, glycogen is the ultimate fuel and is fermented into lactate, supplying ATP. During recovery the lactate is reconverted (through gluconeogenesis) to glycogen and glucose in the liver.
Phosphocreatine is an immediate source of ATP
during active contraction. Heart muscle obtains all
of its ATP from oxidative phosphorylation. The
brain uses only glucose and /3-hydroxybutyrate as
fuels, the latter being important during fasting or
starvation. The brain uses most of its ATP energy
for the active transport of Na + and K" and the
maintenance of the electrical potential of neuronal
membranes. The blood links all of the organs, carrying nutrients, waste products, and hormonal signals between them.
Hormones are chemical messengers (peptides,
amines, or steroids) secreted by certain tissues into
the blood, serving to regulate the activity of other
tissues. They act in a hierarchy of functions. Nerve
impulses stimulate the hypothalamus to send specific hormones to the pituitary gland, stimulating
(or inhibiting) the release of tropic hormones. The
anterior pituitary hormones in turn stimulate
other endocrine glands (thyroid, adrenals, pancreas) to secrete their characteristic hormones,
which in turn stimulate specific target tissues.
The concentration of glucose in the blood is hormonally regulated. Fluctuations in blood glucose
(which is normally about 80 mg/100 mL or 4.5 DIM)
due to dietary uptake or vigorous exercise are
counterbalanced by a variety of hormonally triggered changes in the metabolism of several organs.
Epinephrine prepares the body for increased activity by mobilizing blood glucose from glycogen and
other precursors. Low blood glucose results in the
release of glucagon, which stimulates glucose release from liver glycogen and shifts the fuel metabolism in liver and muscle to fatty acids, sparing
glucose for use by the brain. In prolonged fasting,
triacylglycerols become the principal fuels; the
liver converts the fatty acids to ketone bodies for
export to other tissues, including the brain. High
blood glucose elicits the release of insulin, which
speeds the uptake of glucose by tissues and favors
the storage of fuels as glycogen and triacylglycerols. In untreated diabetes, insulin is either not produced or is not recognized by the tissues, and the
utilization of blood glucose is compromised. When
blood glucose levels are high, glucose is excreted
intact into the urine. Tissues then depend upon
fatty acids for fuel (producing ketone bodies) and
degrade cellular proteins to make glucose from
Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
783
their glucogenic amino acids. Untreated diabetes is
characterized by high glucose levels in the blood
and urine and the production and excretion of ketone bodies.
Hormones act through a small number of fundamentally similar mechanisms. Epinephrine binds
to specific /3-adrenergic receptors on the outer face
of hepatocytes and myocytes. A stimulatory GTPbinding protein (Gs) mediates between the adrenergic receptor and adenylate cyclase on the inner
face of the plasma membrane. When the adrenergic receptor is occupied, adenylate cyclase is activated and converts ATP to cAMP (the second messenger), which then activates the cAMP-dependent
protein kinase. This protein kinase phosphorylates
and activates inactive phosphorylase b kinase,
which in a subsequent step phosphorylates and
activates glycogen phosphorylase. Cyclic nucleotide phosphodiesterase terminates the signal by
converting cAMP to AMP. The cAMP-dependent
protein kinase also phosphorylates and regulates a
number of other enzymes present in target tissues.
(Glucagon acts by an essentially similar mechanism except that the tissue distribution of glucagon receptors is different; this hormone acts primarily on the liver.) This cascade of events, in
which a single molecule of hormone activates a catalyst that in turn activates another catalyst and so
on, results in large signal amplification; this is
characteristic of all hormone-activated systems.
Cyclic GMP acts as the second messenger for other
hormones, by a similar mechanism.
Protein phosphorylation is a universal mechanism for rapid and reversible enzyme regulation.
To reverse the effects of signal-stimulated protein
kinases, cells contain a variety of phosphatases.
These enzymes, too, are subject to regulation by
extracellular and intracellular signals.
The insulin receptor represents a second signaltransducing mechanism. The receptor is an integral protein of the plasma membrane. Binding of
insulin to its extracellular domain activates a tyrosine-specific protein kinase in the receptor's cytosolic domain. This kinase activates several protein
kinases by phosphorylating specific Tyr residues.
The phosphorylated protein kinases bring about
changes in metabolism by phosphorylating additional key enzymes, altering their enzymatic activities.
A third general class of hormone mechanisms
involves the coupling of hormone receptors, via
another group of GTP-binding proteins, to a phospholipase C of the plasma membrane. Hormone
binding activates this enzyme, which hydrolyzes
inositol-containing phospholipids in the plasma
membrane. This generates two second messengers:
diacylglycerol, which activates protein kinase C,
and inositol-l,4,5-trisphosphate (IP3), which
causes the release of Ca 2+ sequestered in the endoplasmic reticulum. Ca 2+ is a common second messenger in hormone-sensitive cells and in neural
signaling; it alters the enzymatic activities of specific protein kinases. Calmodulin is a small Ca2+binding subunit of a number of Ca2+-dependent
enzymes.
The fourth general transduction mechanism
triggered by hormones is the opening of hormonesensitive ion channels. The nicotinic acetylcholine
receptor is a ligand-gated ion channel, which,
when occupied by acetylcholine, allows transmembrane passage of Na + and K+ ions and consequent
depolarization of the target cell. A wave of depolarization sweeps along nerves through the action of
voltage-gated Na + and Ca 2+ ion channels, triggering neurotransmitter release.
A variety of pathological conditions are associated with defects in signal-transduction mechanisms. Some bacterial toxins interfere with signal
transductions. Oncogenes in a cell's DNA permit
uncontrolled cell division, possibly through formation of defective signal-transducing proteins that
are insensitive to modulation by growth factors or
hormonal signals. Tumor promoters also interfere
with cell regulation and growth.
Steroid hormones enter cells and bind to specific
receptor proteins. The hormone-receptor complex
binds specific regions of nuclear DNA called hormone response elements and regulates the expression of nearby genes. Tamoxifen and RU486 are
drugs that act as steroid hormone antagonists.
General Background and History
Nishizuka, Y., Tanaka, C, & Endo, M. (eds)
(1990) The Biology and Medicine of Signal Transduction, Adv. Second Messenger Phosphoprotein
Res., 24.
A collection of papers on receptor-transducer systems and the medical effects of defective signal
transducers.
Further Reading
Molecular Biology of Signal Transduction. (1988)
Cold Spring Harb. Symp. Quant. Biol. 53.
This entire volume is filled with short research and
review papers on a wide variety of signal-transducing systems, from bacteria to humans.
784
Part III Bioenergetics and Metabolism
Sutherland, E.W. (1972) Studies on the mechanisms of hormone action. Science 177, 401-408.
The author's Nobel lecture, describing the classic
experiments on cAMP.
Wilson, J.D. & Foster, D.W. (eds) (1992) Williams
Textbook of Endocrinology, 8th edn, W.B. Saunders
Company, Philadelphia.
Especially relevant are Chapter 1, an introduction
to hormonal regulation; Chapter 3, on the mechanism of action of steroid hormones; and Chapter 4,
on the mechanisms of hormones that act at the cell
surface.
Yalow, R.S. (1978) Radioimmunoassay: a probe for
the fine structure of biologic systems. Science 200,
1236-1245.
A history of the development of radioimmunoassays; the author's Nobel lecture.
Tissue-Specific Metabolism: Division of
Labor
Arias, I.M., Jakoby, W.B., Popper, H., Schachter,
D., & Shafritz, D.A. (eds) (1988) The Liver: Biology
and Pathobiology, 2nd edn, Raven Press, New
York.
An advanced-level text; includes chapters on the
metabolism of carbohydrates, fats, and proteins in
the liver.
Hormones: Communication among Cells and
Tissues
Crapo, L. (1985) Hormones: The Messengers of
Life, W.H. Freeman and Company, New York.
A short, entertaining account of the history and recent state of hormone research.
Snyder, S.H. (1985) The molecular basis of communication between cells. Sci. Am. 253 (October),
132-141.
An introductory-level discussion of the human endocrine system.
Hormonal Regulation of Fuel Metabolism
Harris, R.A. & Crabb, D.W. (1992) Metabolic interrelationships. In Textbook of Biochemistry with
Clinical Correlations, 3rd edn (Devlin, T.M., ed),
pp. 576-606, John Wiley & Sons, Inc., New York.
A description of the metabolic interplay among
human tissues during normal metabolism, and the
effect on tissue-specific energy metabolism of the
stresses of exercise, lactation, diabetes, and renal
disease.
Pilkis, S.J. & Claus, T.H. (1991) Hepatic gluconeogenesis/glycolysis: regulation and structure/function relationships of substrate cycle enzymes.
Annu. Rev. Nutr. 11, 465-515.
A review at the advanced level.
Roach, P.J. (1990) Control of glycogen synthase by
hierarchal protein phosphorylation. FASEB J. 4,
2961-2968.
Phosphorylation of one enzyme at several positions
by several different protein kinases can produce
finely graded changes in enzyme activity.
Molecular Mechanisms of Signal
Transduction
Aaronson, S.A. (1991) Growth factors and cancer.
Science 254, 1146-1153.
A clear description of defects in the signal-transducing mechanisms that regulate cell division,
which result from mutations in the genes for
growth-factor receptors.
Becker, A.B. & Roth, R.A. (1990) Insulin receptor
structure and function in normal and pathological
conditions. Annu. Rev. Med. 41, 99-115.
A brief description of the structure of the receptor
and its gene, and a discussion of the clinical syndromes associated with receptor defects.
Berridge, M.J. (1985) The molecular basis of communication within the cell. Sci. Am. 253 (October),
142-152.
An introduction to the transductions mediated by
adenylate cyclase, guanylate cyclase, and phospholipase C.
Berridge, M.J. & Irvine, R.F. (1989) Inositol phosphates and cell signalling. Nature 341, 197-205.
Not the latest, but one of the best descriptions of the
role of inositol phospholipids in signal transduction.
Brent, G.A., Moore, D.D., & Larsen, P.R. (1991)
Thyroid hormone regulation of gene expression.
Annu. Rev. Physiol. 53, 17-36.
An advanced discussion.
Collins, S., Lohse, M.J., O'Dowd, B., Caron, M.G.,
& Lefkowitz, R.J. (1991) Structure and regulation
of G protein-coupled receptors: the /32-adrenergic
receptor as a model. Vitam. Horm. 46, 1-39.
An advanced discussion.
Fisher, S.K., Heacock, A.M., & Agranoff, B.W.
(1992) Inositol lipids and signal transduction in the
nervous system: an update. J. Neurochem. 58,1838.
A review of inositol phospholipids in signaling, including a good description of the various phosphorylated derivatives of inositol and their functions as
second messengers; advanced level.
Gilman, A.G. (1989) G proteins and regulation of
adenylyl cyclase. JAMA 262, 1819-1825.
Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
Hille, B. (1991) Ionic Channels of Excitable Membranes, 2nd edn, Sinauer Associates, Sunderland,
MA.
Very broad coverage, at an intermediate level.
Hollenberg, M.D. (1991) Structure-activity relationships for transmembrane signaling: the receptor's turn. FASEB J. 5, 178-186.
A description of how information about the amino
acid sequences of receptors, derived from cloning
receptor genes, can be used to discover structural
bases for receptor interactions with ligands, G proteins, and other elements of a transducing system.
Kennelly, P.J. & Krebs, E.G. (1991) Consensus
sequences as substrate specificity determinants for
protein kinases and protein phosphatases. J. BioL
Chem. 266, 15555-15558.
A concise summary of the sequence specificity of
protein kinases.
Krebs, E.G. (1989) Role of the cyclic AMPdependent protein kinase in signal transduction.
JAMA 262, 1815-1818.
A clear account of the research on protein kinase A
and its history.
Linder, M.E. & Gilman, A.G. (1992) G proteins.
Sci. Am. 267 (July), 56-65.
An introductory level description of the discovery
and functions of GTP-binding proteins.
785
Rasmussen, H. (1989) The cycling of calcium as an
intracellular messenger. Sci. Am. 261 (October),
66-73.
An introduction to the role ofCa2+ as a second messenger.
Snyder, S.H. & Bredt, D.S. (1992) Biological roJes
of nitric oxide. Sci. Am. 266 (May), 68-77.
An intermediate-level review of the role of NO as a
second messenger.
Taylor, S.S., Buechler, J.A., & Yonemoto, W.
(1990) cAMP-dependent protein kinase: framework
for a diverse family of regulatory enzymes. Annu.
Rev. Biochem. 59, 971-1005.
An advanced review of the structure and function of
protein kinase A and a comparison of its activation
mechanism and catalytic mechanism with those of
other protein kinases.
Ulmann, A., Teutsch, G., & Philibert, D. (1990)
RU 486. Sci. Am. 262 (June), 42-48.
The effects of this steroid antagonist, the "morningafter pill," on the female reproductive system; an
introduction.
Ullrich, A. & Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity.
Cell 61, 203-212.
A review of the common structural and functional
features of receptors in the insulin receptor family.
O'Malley, B.W., Tsai, S.Y., Bagchi, M., Weigel,
N.L., Schrader, W.T., & Tsai, M.-J. (1991) Molecular mechanism of action of a steroid hormone receptor. Recent Prog. Horm. Res. 47, 1-26.
A brief history of the discovery of steroid hormone
receptors and their genes, and a review of the effects
of the hormone-receptor complex on mRNA and
protein synthesis in vitro.
Problems
1. ATP and Phosphocreatine as Sources of Energy
for Muscle In contracting skeletal muscle, the
concentration of phosphocreatine drops while the
concentration of ATP remains fairly constant. Explain how this happens.
In a classic experiment, Robert Davies found
that if the muscle is first treated with l-fluoro-2,4dinitrobenzene (see Fig. 5-14), the concentration of
ATP in the muscle declines rapidly, whereas the
concentration of phosphocreatine remains unchanged during a series of contractions. Suggest an
explanation.
2. Metabolism of Glutamate in the Brain Glutamate in the blood flowing into the brain is transformed into glutamine, which appears in the blood
leaving the brain. What is accomplished by this
metabolic conversion? How does it take place? Actually, the brain can generate more glutamine
than can be made from the glutamate entering in
the blood. How does this extra glutamine arise?
(Hint: You may want to review amino acid catabolism in Chapter 17. Recall that NH 3 is very toxic to
the brain.)
786
Part III Bioenergetics and Metabolism
3. Absence of Glycerol Kinase in Adipose Tissue
Glycerol-3-phosphate is a key intermediate in the
biosynthesis of triacylglycerols. Adipocytes, which
are specialized for the synthesis and degradation
of triacylglycerols, cannot directly use glycerol because they lack glycerol kinase, which catalyzes
the reaction
Glycerol + ATP
glycerol-3-phosphate + ADP
How does adipose tissue obtain the glycerol-3phosphate necessary for triacylglycerol synthesis?
Explain.
4. Hyperglycemia in Patients with Acute Pancreatitis Patients with acute pancreatitis are treated by
withholding protein from the diet and by intravenous administration of glucose-saline solution.
What is the biochemical basis for these measures?
Patients undergoing this treatment commonly experience hyperglycemia. Why?
5. Oxygen Consumption during Exercise A sedentary adult consumes about 0.05 L of O2 during a
10 s period. A sprinter, running a 100 m race, consumes about 1 L of O2 during the same time period.
After finishing the race, the sprinter will continue
to breathe at an elevated but declining rate for
some minutes, consuming an extra 4 L of O2 above
the amount consumed by the sedentary individual.
(a) Why do the O2 needs increase dramatically
during the sprint?
(b) Why do the O2 demands remain high after
the sprint is completed?
6. Thiamin Deficiency and Brain Function Individuals with thiamin deficiency display a number
of characteristic neurological signs: loss of reflexes,
anxiety, and mental confusion. Suggest a reason
why thiamin deficiency is manifested by changes
in brain function.
7. Significance of Hormone Concentration Under
normal conditions, the human adrenal medulla
secretes epinephrine (C9H13NO3) at a rate sufficient to maintain a concentration of 10 ~10 M in the
circulating blood. To appreciate what that concentration means, calculate the diameter of a round
swimming pool, with a water depth of 2 m, that
would be needed to dissolve 1 g (about 1 teaspoon)
of epinephrine to a concentration equal to that in
blood.
8. Regulation of Hormone Levels in the Blood The
half-life of most hormones in the blood is relatively
short. For example, if radioactively labeled insulin
is injected into an animal, one can determine that
within 30 min half the hormone has disappeared
from the blood.
(a) What is the importance of the relatively
rapid inactivation of circulating hormones?
(b) In view of this rapid inactivation, how can
the circulating hormone level be kept constant
under normal conditions?
(c) In what ways can the organism make possible rapid changes in the level of circulating hormones?
9. Water-Soluble versus Lipid-Soluble Hormones
On the basis of their physical properties, hormones
fall into one of two categories: those that are very
soluble in water but relatively insoluble in lipids
(e.g., epinephrine) and those that are relatively
insoluble in water but highly soluble in lipids (e.g.,
steroid hormones). In their role as regulators of
cellular activity, most water-soluble hormones do
not penetrate into the interior of their target cells.
The lipid-soluble hormones, by contrast, do penetrate into their target cells and ultimately act in
the nucleus. What is the correlation between solubility, the location of receptors, and the mode of
action of the two classes of hormones?
10. Hormone Experiments in Cell-Free Systems In
the 1950s, Earl Sutherland and his colleagues carried out pioneering experiments to elucidate the
mechanism of action of epinephrine and glucagon.
In the light of our current understanding of hormone action as described in this chapter, interpret
each of the experiments described below. Identify
the components and indicate the significance of the
results.
(a) The addition of epinephrine to a homogenate
or broken-cell preparation of normal liver resulted
in an increase in the activity of glycogen phosphorylase. However, if the homogenate was first centrifuged at a high speed and epinephrine or glucagon was added to the clear supernatant fraction
containing phosphorylase, no increase in phosphorylase activity was observed.
(b) When the particulate fraction sedimented
from a liver homogenate by centrifugation was separated and treated with epinephrine, a new substance was produced. This substance was isolated
and purified. Unlike epinephrine, this substance
activated glycogen phosphorylase when added to
the clear supernatant fraction of the homogenate.
(c) The substance obtained from the particulate
fraction was heat-stable; that is, heat treatment
did not prevent its capacity to activate phosphorylase. (Hint: Would this be the case if the substance
were a protein?) The substance appeared nearly
identical to a compound obtained when pure ATP
was treated with barium hydroxide. (Figure 12-6
will be helpful.)
11. Effect of Dibutyryl-cAMP versus cAMP on Intact Cells The physiological effects of the hormone
epinephrine should in principle be mimicked by
the addition of cAMP to the target cells. In practice, the addition of cAMP to intact target cells elicits only a minimal physiological response. Why?
When the structurally related derivative dibutyryl-cAMP (shown below) is added to intact cells,
the expected physiological responses can readily be
seen. Explain the basis for the difference in cellu-
Chapter 22 Integration and Hormonal Regulation of Mammalian Metabolism
lar response to these two substances. Dibutyryl
cAMP is a widely used derivative in studies of
cAMP function.
NH—C-CH 2 —CH2 - C H 3
O-CH 2
O=P
O"
O
O-C-CH2-CH2-CH3
O
Dibutyryl-cAMP
12. Effect of Cholera Toxin on Adenylate Cyclase
The gram-negative bacterium Vibrio cholerae produces a protein, cholera toxin (Mr 90,000), responsible for the characteristic symptoms of cholera:
extensive loss of body water and Na + through continuous, debilitating diarrhea. If body fluids and
Na + are not replaced, severe dehydration will
occur; untreated, the disease is often fatal. When
the cholera toxin gains access to the human intestinal tract it binds tightly to specific sites in the
plasma membrane of the epithelial cells lining the
small intestine, causing adenylate cyclase to undergo activation that persists for hours or days.
(a) What is the effect of cholera toxin on the
level of cAMP in the intestinal cells?
(b) Based on the information above, can you
suggest how cAMP normally functions in intestinal
epithelial cells?
(c) Suggest a possible treatment for cholera.
13. Metabolic Differences in Muscle and Liver in a
'Tight or Flight" Situation During a "fight or
flight" situation, the release of epinephrine promotes glycogen breakdown in the liver, heart, and
skeletal muscle. The end product of glycogen
breakdown in the liver is glucose. In contrast, the
end product in skeletal muscle is pyruvate.
787
(a) Why are different products of glycogen
breakdown observed in the two tissues?
(b) What is the advantage to the organism during a "fight or flight" condition of having these specific glycogen breakdown routes?
14. Excessive Amounts of Insulin Secretion: Hyperinsulinism Certain malignant tumors of the pancreas cause excessive production of insulin by the p
cells. Affected individuals exhibit shaking and
trembling, weakness and fatigue, sweating, and
hunger. If this condition is prolonged, brain damage occurs.
(a) What is the effect of hyperinsulinism on the
metabolism of carbohydrate, amino acids, and lipids by the liver?
(b) What are the causes of the observed symptoms? Suggest why this condition, if prolonged,
leads to brain damage.
15. Thermogenesis Caused by Thyroid Hormones
Thyroid hormones are intimately involved in regulating the basal metabolic rate. Liver tissue of animals given excess thyroxine shows an increased
rate of O2 consumption and increased heat output
(thermogenesis), but the ATP concentration in the
tissue is normal. Different explanations have been
offered for the thermogenic effect of thyroxine. One
is that excess thyroid hormone causes uncoupling
of oxidative phosphorylation in mitochondria. How
could such an effect account for the observations?
Another explanation suggests that the thermogenesis is due to an increased rate of ATP utilization
by the thyroid-stimulated tissue. Is this a reasonable explanation? Why?
16. Function of Prohormones What are the possible advantages in the synthesis of hormones as
prohormones or preprohormones?
17. Action of Aminophylline
Aminophylline, a
purine derivative resembling theophylline of tea, is
often administered together with epinephrine to
individuals with acute asthma. What is the purpose and biochemical basis for this treatment?
C H A P T E R
Regulation of Gene Expression
Of the 4,000 genes in the typical bacterial genome or the estimated
100,000 genes in the human genome, only a fraction are expressed at
any given time. Some gene products have functions that mandate their
presence in very large amounts. The elongation factors required for
protein synthesis, for example, are among the most abundant proteins
in bacteria. Other gene products are needed in much smaller amounts;
for instance, a cell may contain only a few molecules of the enzymes
that repair rare DNA lesions. Requirements for a given gene product
may also change with time. The need for enzymes in certain metabolic
pathways may wax or wane as food sources change or are depleted.
During development in a multicellular eukaryote, some proteins that
influence cellular differentiation are present for only a brief time in a
small subset of an organism's cells. The specialization of some cells for
particular functions can also dramatically affect the need for various
gene products, one example being the uniquely high concentration of
hemoglobin in erythrocytes.
The regulation of gene expression is a critical component in regulating cellular metabolism and in orchestrating and maintaining the
structural and functional differences that exist in cells during development. Given the high energetic cost of protein synthesis, regulation of
gene expression is essential if the cell is to make optimal use of available energy.
Regulating the concentration of a cellular protein involves a delicate balance of many processes. There are at least six potential points
at which the amount of protein can be regulated (Fig. 27-1): synthesis
of the primary RNA transcript, posttranscriptional processing of
mRNA, mRNA degradation, protein synthesis (translation), posttranslational modification of proteins, and protein degradation. The concentration of a given protein is controlled by regulatory mechanisms at
any or all of these points. Some of these mechanisms have been examined in previous chapters. Posttranscriptional modification of mRNAs
by processes such as differential splicing (p. 873) or RNA editing (see
Box 26-1) can affect which proteins are produced from an mRNA transcript and in what amounts. A variety of sequences can affect the rate
at which an mRNA is degraded (p. 880). Many factors that affect the
rate at which an mRNA is translated into a protein, as well as the
posttranslational modification and eventual degradation of that protein, were described in Chapter 26.
Our primary focus in this chapter is the regulation of transcription
initiation (although some aspects of the regulation of translation will
942
Part IV Information Pathways
Gene
DNA'
Transcription
Primary transcript *V/V
V/V
Posttranscriptional
processing
j
Nucleotides
mRNA degradation
Mature mRNA
Translation
Protein
(inactive)
Amino acids
Posttranslational
processing
Protein degradation
Modified
protein
(active)
Figure 27-1 Six processes that affect the steadystate concentration of a protein. Each of these processes is a potential point of regulation.
be described). Of all the processes illustrated in Figure 27-1, regulation at the level of transcription initiation is the best documented and
may be the most common. At least one important reason is clear: as for
all biosynthetic pathways, the most efficient place for regulation is the
first reaction in the pathway. In this way, unnecessary biosynthesis
can be halted before energy is invested. Transcription initiation also is
an excellent point at which to coordinate the regulation of multiple
genes whose products have interdependent activities. For example,
when DNA is heavily damaged, bacterial cells require a coordinated
increase in the levels of many enzymes involved in DNA repair.
Perhaps the most sophisticated form of coordination occurs in the
complex regulatory circuits that guide the development of multicellular
eukaryotes.
In this chapter, we first describe the interactions between proteins
and DNA that are the key to transcriptional regulation. Specific proteins that regulate the expression of specific genes will then be discussed, first for prokaryotes and then for eukaryotes. In the course of
this discussion we will examine several different mechanisms by which
cells regulate gene expression and coordinate the expression of multiple genes.
Chapter 27 Regulation of Gene Expression
943
Gene Regulation: Principles and Proteins
Just as the cellular requirements for different proteins vary, the mechanisms by which their respective genes are regulated also vary. The
degree and type of regulation naturally reflect the function of the protein product of the gene. Some gene products are required all the time
and their genes are expressed at a more or less constant level in virtually all the cells of a species or organism. Many of the genes for enzymes that catalyze steps in central metabolic pathways such as the
citric acid cycle fall into this category. These genes are often referred to
as housekeeping genes. Constant, seemingly unregulated expression of a gene is called constitutive gene expression. The amounts of
other gene products rise and fall in response to molecular signals. Gene
products that increase in concentration under prescribed molecular
circumstances are referred to as inducible, and the process of increasing the expression of the gene is called induction. The expression of
many genes encoding DNA repair enzymes, for example, is induced in
response to high levels of DNA damage. Conversely, gene products
that decrease in concentration in response to a molecular signal are
referred to as repressible, and the decrease in gene expression is called
repression. For example, the presence of ample supplies of the amino
acid tryptophan leads to repression of the genes for the enzymes catalyzing tryptophan biosynthesis in bacteria.
Transcription is mediated and regulated by protein-DNA interactions. The central component is RNA polymerase, an enzyme described
in some detail in Chapter 25. We begin here with a further description
of RNA polymerase from the standpoint of regulation, then proceed to
a general description of the proteins that modulate the activity of RNA
polymerase. Finally we discuss the molecular basis for the recognition
of specific DNA sequences by DNA-binding proteins.
The Activity of RNA Polymerase Is Regulated
RNA polymerases bind to DNA and initiate transcription at specific
sites in the DNA called promoters (Chapter 25). Promoters generally
are found very near the position where RNA synthesis begins on the
DNA template. The regulation of transcription initiation is, in effect,
regulation of the interaction of RNA polymerase with its promoter.
Promoters vary considerably in their nucleotide sequence, and this
affects the binding affinity of RNA polymerases. The binding affinity in
turn affects the frequency of transcription initiation. In E. coli, some
genes are transcribed once each second whereas others are transcribed
less than once per cell generation. Much of this variation is accounted
for simply by differences in promoter sequences. In the absence of regulatory proteins, differences in the sequences of two promoters may
affect the frequency of transcription initiation by factors of 1,000 or
more. Recall (see Fig. 25—5) that E. coli promoters have a consensus
sequence (Fig. 27-2). Promoters that exactly match the consensus se-
-35 region
-10 region
RNA start site
DNA 5'
m
R
N
A\/\/\/V
Figure 27-2 Consensus sequence for many E. coli
promoters. N indicates any nucleotide. Most base
substitutions in the -10 and -35 regions have a
negative effect on promoter function. (Recall from
Chapter 25 that by convention, DNA sequences are
shown as they occur on the coding (nontemplate)
strand.)
944
Part IV Information Pathways
quence generally have the highest affinity for RNA polymerase and the
highest frequency of transcription initiation. Mutations that change a
consensus base pair to a nonconsensus pair generally decrease promoter function: mutations that change a nonconsensus base pair to a
consensus pair usually enhance promoter function.
Although housekeeping genes are expressed constitutively, the
proteins they encode are present in widely varying amounts. For these
genes the RNA polymerase-promoter interaction is the only factor affecting transcription initiation, and differences in promoter sequences
allow the cell to maintain the required level of each housekeeping
protein.
Transcription initiation at the promoters of many genes that do not
fall in the housekeeping category is further regulated in response to
molecular signals. These promoters have a basal rate of transcription
initiation (determined by the promoter sequence), superimposed on
which is regulation mediated by several types of regulatory proteins.
These proteins affect the interaction between RNA polymerase and the
promoters.
Transcription Initiation Is Regulated by Proteins
Binding to or near Promoters
At least three types of proteins regulate transcription initiation by
RNA polymerase: (1) specificity factors alter the specificity of RNA
polymerase for a given promoter or set of promoters; (2) repressors
bind to a promoter, blocking access of RNA polymerase to the promoter; (3) activators bind near a promoter, enhancing the RNApromoter interaction.
We encountered prokaryotic specificity factors in Chapter 25, although they were not given that name. The <x subunit (Mr 70,000)
called a70 of the E. coli RNA polymerase holoenzyme is a prototypical
specificity factor that mediates specific promoter recognition and binding. Under some conditions, notably when the bacteria are subjected to
heat stress, cr70 is replaced with another specificity factor (Mr 32,000)
called a32 (p. 863). When bound to a32, RNA polymerase does not bind
to the standard E. coli promoters (Fig. 27-2), but instead is directed to
a specialized set of promoters with the sequence structure shown in
Figure 27-3. The promoters control the expression of a set of genes
that make up the heat-shock response. Altering the polymerase to direct it to different promoters is one mechanism by which a set of related genes can be coordinately regulated. Other mechanisms will be
encountered throughout this chapter.
Figure 27—3 Consensus sequence for promoters
that regulate the expression of genes involved in
the heat-shock response in E. coli. This system responds to temperature increases as well as some
other environmental stresses, and it involves the
induction of a set of proteins. Binding of RNA polymerase to heat-shock promoters is mediated by a
specialized a subunit of the enzyme called cr32,
which replaces <x70.
LRNA start site
DNA 5
TNTCNCCCTTGAA
N _
13 15
CCCCATTTA N7
mRNA
Repressors bind to specific sites in the DNA. In prokaryotes, the
binding sites for repressors are called operators. Operator sites are
generally near and often overlap the promoter so that RNA polymerase
binding, or its movement along the DNA after binding, is blocked
whenever the repressor is present. Regulation by means of a repressor
Negative regulation
Positive regulation
(bound repressor inhibits transcription)
(bound activator facilitates transcription)
(c)
(a)
RNA polymerase
Operator
Molecular signal
( ^ ) causes dissociation
of regulatory protein
from DNA
DNA
Promoter
mRNA
mRNA
(d)
(b)
Molecular signal
( ^ ) causes binding
of regulatory protein
to DNA
mRNA
mRNA
protein that binds to DNA and blocks transcription is referred to as
negative regulation. Repressor binding is regulated by a molecular
signal, usually a specific small molecule that binds to and induces a
conformational change in the repressor. The interaction between repressor and signal molecule may lead to either an increase or a decrease in transcription. In some cases the conformational change results in dissociation of a DNA-bound repressor from the operator (Fig.
27-4a). Transcription initiation can then proceed unhindered. In other
cases the interaction between an inactive repressor and the signal molecule causes the repressor to bind to the operator (Fig. 27-4b).
Activators provide a molecular counterpoint to repressors. Regulation mediated by an activator is called positive regulation. Activators bind to sites adjacent to a promoter and enhance the binding and
activity of RNA polymerase at that promoter. The binding sites for
activators are often found adjacent to promoters that are normally
bound weakly or not at all by RNA polymerase. Transcription at these
genes is therefore often negligible in the absence of activator. Sometimes the activator is normally bound to DNA and dissociates when it
binds to the signal molecule, often a specific small molecule or another
protein (Fig. 27-4c). When bound to the DNA, the activator protein
facilitates RNA polymerase binding and increases the rate of transcription initiation. In other cases the activator is not bound to the
DNA until it also binds to a molecular signal (Fig. 27-4d). Positive
regulation is particularly common in eukaryotes, as we shall see. We
now turn to a fundamental unit of gene expression, the study of which
gave rise to much of our current understanding of the regulation of
gene expression.
Figure 27-4 Common patterns of regulation of
transcription initiation. Two types of negative regulation are illustrated, (a) The repressor (red) is
bound to the operator in the absence of the molecular signal; the signal causes dissociation of the repressor to permit transcription, (b) The repressor is
bound in the presence of the signal; the repressor
dissociates and transcription ensues when the signal is removed. Positive regulation is mediated by
gene activators, (c) The activator (green) binds in
the absence of the molecular signal and transcription proceeds; the activator dissociates and transcription is inhibited when the signal is added,
(d) The activator binds in the presence of the signal; it dissociates only when the signal is removed.
Note that "positive" and "negative" regulation are
defined by the type of regulatory protein involved.
In either case the addition of the molecular signal
may increase or decrease transcription, depending
on the effect of the signal on the regulatory protein.
Part IV Information Pathways
946
Figure 27—5 An operon. Genes A, B, and C are
transcribed on one polycistronic mRNA. Typical
regulatory sequences include binding sites for proteins that either activate or repress transcription
from the promoter.
Repressor
binding site
(operator)
Activator
binding site
I
DNA
\
| Promoter
Wllllllillll
/
\ Regulatory sequences1
c
A
J
V
V
Genes transcribed as a unit
Many Prokaryotic Genes Are Regulated
in Units Called Operons
Lactose
Galactoside permease
Outside
H
H
OH
OH
Lactose
/3-galacto.sidase
O-CH 2
?/4~°\?H
H
OH
Galactose
H
OH
Glucose
Bacteria have a simple general mechanism for coordinating the regulation of genes whose products are involved in related processes: the
genes are clustered on the chromosome and transcribed together. Most
prokaryotic mRNAs are polycistronic. The single promoter required to
initiate transcription of the cluster is the point where expression of all
of the genes is regulated. The gene cluster, the promoter, and additional sequences that function in regulation are together called an operon (Fig. 27-5). Operons that include 2 to 6 genes transcribed as a
unit are common; some operons contain 20 or more genes.
Many of the principles guiding the regulation of gene expression in
bacteria were defined by studies of the regulation of lactose metabolism in E. coli. The disaccharide lactose can be used as the sole carbon
source for the growth of E. coli. In 1960, Frangois Jacob and Jacques
Monod published a short paper in the Proceedings of the French Academy of Sciences demonstrating that two genes involved in lactose metabolism were coordinately regulated by a genetic element located adjacent to them. The genes were those for /3-galactosidase, which cleaves
lactose to galactose and glucose, and galactoside permease, which
transports lactose into the cell (Fig. 27-6). The terms operon and operator were first introduced in this paper. The operon model that evolved
from this and subsequent studies permitted biochemists to think about
gene regulation in molecular terms for the first time.
The lac Operon Is Subject to Negative Regulation
The model for regulation of the lactose (lac) operon deduced from these
studies is shown in Figure 27-7; it follows the pattern outlined in
Figure 27-4a. In addition to the genes for /3-galactosidase (Z) and galactoside permease (Y), the operon includes a gene for thiogalactoside
transacetylase (A), whose physiological function is unknown. Each of
the three genes is preceded by translational signals (not shown in Fig.
27-7) to guide ribosome binding and protein synthesis (Chapter 26). In
Figure 27-6 The activities of galactoside permease
and /3-galactosidase in lactose metabolism in
E. coli. The conversion of lactose to allolactose by
transglycosylation is a minor reaction catalyzed by
/3-galactosidase.
947
Chapter 27 Regulation of Gene Expression
Figure 27-7 The lac operon in the repressed
state. The I gene encodes the Lac repressor. The
lac Z, Y, and A genes encode /3-galactosidase, galactoside permease, and transacetylase, respectively.
The P and 0 sites are the promoter and operator
for the lac genes, respectively. The Pi site is the
promoter for the I gene.
?
Repressor ^{5^§c f~
mRNA\/\/\/
DNA
Px
the absence of the substrate lactose, the lac operon genes are repressed, and j3-galactosidase is present in only a few copies (a few molecules) per cell. Jacob and Monod found that mutations in the operator
or in another gene called I led to constitutive synthesis of the lac operon gene products. When the I gene was defective, repression could be
restored by introducing a functional I gene to the cell on another DNA
molecule. This showed that the I gene encoded a diffusible molecule
that caused gene repression; the molecule was later shown to be a
protein, now called the Lac repressor. Repression is not absolute. Even
in the repressed state each cell has a few copies of /3-galactosidase and
galactoside permease, presumably synthesized on the rare occasions
when the repressor briefly dissociates from its DNA binding site (the
operator).
When cells are provided with lactose, the lac operon is induced. An
inducer molecule binds to a specific site on the repressor causing a
conformational change in the repressor that results in its dissociation
from the operator (Fig. 27-8). The inducer in this system is not lactose
itself but an isomer of lactose called allolactose (Fig. 27-6). Lactose
entering the E. coli cell is converted to allolactose in a reaction catalyzed by the few copies of /3-galactosidase in the cell. Allolactose then
binds to the Lac repressor. After the repressor dissociates, the lac operon genes are expressed and the concentration of /3-galactosidase increases by a factor of 1,000.
Jacques Monod
Frangois Jacob
DNA
Allolactose
(or IPTG)
Y
mRNA
A
Figure 27-8 Induction of the lac operon in response to a molecular signal. Binding of allolactose
to the Lac repressor causes a conformational
change. The repressor dissociates from the operator, allowing transcription to proceed. Other
/3-galactosides, such as isopropylthiogalactoside
(IPTG), can also act as inducers.
Part IV Information Pathways
948
Several /3-galactosides structurally related to allolactose are inducers of, but not substrates for, /3-galactosidase, and some are substrates
but not inducers. One particularly effective and nonmetabolizable inducer of the lac operon often used experimentally is isopropylthiogalactoside (IPTG). Such nonmetabolized inducers permit the separation of the physiological function of lactose as a carbon source for
growth from its function in the regulation of gene expression.
Many operons are now known in bacteria and a few have been
found in lower eukaryotes. The mechanisms by which they are regulated can vary significantly from the simple model presented in Figure
27-7. Research has shown that even the lac operon is more complex
than indicated here, with an activator protein also contributing to the
overall scheme. The regulation of several well-studied bacterial operons, including lac, is described in more detail later in this chapter. We
now consider the critical molecular interactions between DNA-binding
proteins (e.g., repressors and activators) and the specific DNA sequences to which they bind.
CH2OH
H
OH
Isopropylthiogalactoside
(IPTG)
Regulatory Proteins Have Discrete DNA-Binding Domains
Figure 27—9 Functional groups on DNA base pairs
in the major groove of DNA. The groups that can
be used for base-pair recognition are shown in red
for all four base pairs.
Major groove
Regulatory proteins generally bind to specific DNA sequences. They
also bind to nonspecific DNA, but their affinity for their target sequences is generally 105 to 107 times higher. The molecular basis for
this discrimination has been the subject of intensive investigation. A
general conclusion is that regulatory proteins usually have discrete
DNA-binding domains. In addition, the substructures within these
domains that actually come in contact with the DNA fall into one of
a rather small group of recognizable and characteristic structural
motifs.
Before examining these protein structures, it is useful to consider
the recognition surfaces on the DNA with which regulatory proteins
must interact. Most of the groups that differ from one base to another
and can therefore permit discrimination between base pairs are hydrogen-bond donor and acceptor groups exposed in the major DNA
groove (Fig. 27-9). Most of the protein-DNA contacts that impart
specificity are therefore hydrogen bonds. One notable exception is a
nonpolar surface near C-5 of pyrimidines, where thymine is readily
distinguished from cytosine by virtue of thymine's protruding methyl
group (Fig. 27-9). Protein-DNA contacts are also possible in the minor
groove of the DNA, but the hydrogen-bonding patterns here generally
do not allow ready discrimination between different base pairs.
Major groove
Major groove
Major groove
Minor groove
Minor groove
H
Minor groove
msmm
Minor groove
Chapter 27 Regulation of Gene Expression
H O
H 0
I II
R-N-C-C-R'
Glutamine
(or asparagine)
R-N-C-C-R'
Arginine
H
JL
—H
CH 3
Thymine: Adenine
Cytosine: Guanine
As for the regulatory proteins themselves, the amino acid residues
whose side chains are most often found hydrogen-bonded to bases in
the DNA include Asn, Gin, Glu, Lys, and Arg. Is there a simple "recognition code" in which an amino acid is always paired with a certain
base? The two hydrogen bonds that can form between Gin or Asn and
the N 6 and N-7 positions of adenine (Fig. 27-10) constitute a pattern
that cannot form with any other base. An Arg residue can similarly
form two hydrogen bonds to both N-7 and O6 of guanine (Fig. 27-10).
However, examination of the structures of many DNA-binding proteins has shown that there are multiple ways for a protein to recognize
each base pair, and no simple code exists. The Gln-adenine interaction
specifies A=T base pairs in some cases, whereas a van der Waals
pocket for the methyl group of thymine is the mechanism used to recognize A=T base pairs in other proteins. It is not yet possible to examine
the structure of a DNA-binding protein and infer the sequence of the
DNA to which it binds.
The DNA-binding domains of regulatory proteins tend to be small
(60 to 90 amino acid residues). Only a small subset of the amino acids
within these domains actually contact the DNA, and the structure of
the protein in the region where these amino acids occur is not random.
Two structural motifs that play a major role in DNA binding have been
found in numerous regulatory proteins: the helix-turn-helix motif
and the zinc finger. Other DNA-binding motifs exist in some proteins,
but the discussion here focuses on these well-studied examples.
The Helix-Turn-Helix This DNA-binding motif was the first to be
studied in detail. It is the physical basis for protein-DNA interactions
for many prokaryotic regulatory proteins. Closely related DNA-binding motifs also occur in some eukaryotic regulatory proteins. The helixturn-helix motif consists of two short a-helical segments 7 to 9 amino
acid residues long, separated by a /3 turn (about 20 amino acids total).
This structure generally is not stable by itself, but it represents the
reactive portion of the larger DNA-binding domain. One of the two a
helices is referred to as the recognition helix, because it usually contains many of the amino acids that interact with the DNA; this helix is
positioned in the major groove. The Cro repressor protein from bacteriophage 434 (a close relative of bacteriophage A) provides a good examDie (Fie. 27-11).
949
Figure 27-10 Two examples of specific amino
acid—base pair interactions that have been observed
in the structures of DNA-bound regulatory proteins.
950
Part IV Information Pathways
i
4
(b)
•
;
«
#
•
•
#
1
1
Figure 27—11 The Cro repressor of bacteriophage
434 and its interaction with DNA. Each subunit of
this dimeric protein contains 71 amino acids. It is
presented as a ribbon in (a) and (b), alone and
complexed with its specific DNA binding site. The
two subunits are shown in gray and light blue, except for the helix-turn-helix motif in each which is
shown in red and yellow. The red helices are the
recognition helices, which are positioned in adjacent
major grooves of the DNA as seen in (b). The interactions between protein and DNA that allow this
repressor to discriminate between its specific DNA
binding site (shown here) and other DNA sequences
are illustrated in (c) and (d). The protein subunits
are again shown in gray and light blue; chemical
groups on both the DNA and protein that interact
through hydrogen bonds or van der Waals (hydrophobic) interactions are highlighted in red and orange, respectively. Discrimination is mediated by
(d)
interactions between each protein subunit and four
bases (the DNA binding site is a palindrome, and
the interactions are the same for both subunits).
One set of hydrogen bonds is formed between a Gin
residue and the N6 and N-7 of an adenine (see Fig.
27—10); another hydrogen bond is formed between
the O6 of a guanine and another Gin. In addition,
van der Waals pockets on each subunit bind to the
C-5 methyl groups of two adjacent thymines. The
complementary interacting groups are evident in
(c), and the complex is shown in (d). Many nonspecific contacts (not shown) also exist between protein
and DNA in this complex. These do not contribute
to discrimination between DNA sequences, but do
contribute to the overall DNA-binding affinity. An
interesting feature of this structure is that the
DNA is bent slightly when it is bound. This occurs
in the binding of many proteins to DNA (see Fig.
27-16.)
Chapter 27 Regulation of Gene Expression
T/ie Zinc Finger Zinc fingers consist of about 30 amino acid residues;
four of the residues, either four Cys or two Cys and two His, coordinate
a single Zn 2+ atom (Fig. 27-12). This structural motif is found in many
eukaryotic DNA-binding proteins, with several often present in a single protein. There are few, if any, known examples among prokaryotic
proteins. Bacteriophage T4 has a protein, the gene 32 protein, that
binds single-stranded DNA. It binds a single zinc atom within a structure that may be similar to a zinc finger. An apparent record is held by
a DNA-binding protein derived from the frog Xenopus, which has 37
zinc fingers. The precise manner in which proteins containing zinc fingers bind to DNA may vary from one protein to the next. In some cases
these structures contain the amino acid residues that are involved in
sequence discrimination; in other cases the zinc fingers appear to bind
DNA nonspecifically, and the amino acids required for specificity are
found elsewhere in the protein. The interaction of three zinc fingers
(derived from a mouse regulatory protein called Zif 268) with DNA is
shown in Figure 27-12b. It should be noted that some regulatory proteins contain zinc bound within structures that are distinct from the
zinc finger.
Regulatory Proteins Also Interact with Other Proteins
Regulatory proteins generally contain additional domains that are involved in interactions with RNA polymerase, other regulatory proteins, or additional copies of the same regulatory protein (Fig. 27-13).
The DNA binding sites for regulatory proteins are generally inverted
repeats of a short DNA sequence (a palindrome) at which two or four
copies of a regulatory protein bind cooperatively, as in Figures 27-11
and 27-13.
The Lac repressor is a tetramer of identical subunits (Mr 37,000). A
wild-type E. coli cell generally contains about ten copies of Lac repressor. The i gene is transcribed from its own promoter independently of
the lac operon genes (Fig. 27-7). The repressor binds to a palindromic
operator sequence that spans 22 base pairs within the larger regula-
951
Figure 27-12 Zinc fingers, (a) A ribbon representation of a single zinc finger derived from the regulatory protein Zif 268. The zinc atom is in orange
and the amino acid residues that coordinate it (two
His and two Cys) are shown in red. (b) Three zinc
fingers (light blue and gray) from Zif 268 are
shown complexed with DNA. The zinc atoms are
again shown in orange.
1
1
1
1
Figure 27-13 The bacteriophage A repressor
bound to DNA. The two identical subunits of the
dimeric protein are shown in gray and light blue.
RNA
polymerase
(a)
araO2
DNA
CAP binding site
aral
araBAD
araC
ara Oi
r
BAD
araC mRNA
araOo
(b)
araC
araOx
AraC
'proteins
BAD
binding
site
(c)
RNA
polymerase
araC
araBAD
araO2
Figure 27-20 Regulation of the ara operon.
(a) When AraC protein is depleted, the araC gene
is transcribed from its own promoter, (b) When
arabinose levels are low and glucose levels high,
AraC protein binds to both aral and araO2 and
brings these sites together to form a DNA loop.
The operon is repressed in this state. AraC protein
also binds to araOi, repressing further synthesis of
AraC. (c) When arabinose is present and glucose
concentration is low, AraC protein binds arabinose
and changes conformation to become an activator.
The DNA loop is opened, and the AraC protein
acts in concert with CAP-cAMP to facilitate
transcription.
aral
CAP
•
binding
site
Arabinose
BAD
/\/\/\/\/v
araBAD mRNA
Under these conditions, the AraC protein bound to araO2 and that
bound to aral bind to each other, forming a DNA loop of about 210 base
pairs. In this configuration the system represses transcription from
the promoter for the araBAD genes (Fig. 27-20b). (2) Glucose is not
present (or is at low levels) but arabinose is available. Under these
conditions, CAP-cAMP becomes abundant and binds to its site adjacent to aral. Arabinose also binds to the AraC protein, altering its
conformation. The DNA loop is opened, and the AraC protein bound at
aral now becomes an activator, acting in concert with CAP-cAMP to
induce transcription of the araBAD genes (Fig. 27-20c). (3) Arabinose
and glucose are both abundant. (4) Arabinose and glucose are both
absent. For both (3) and (4), the status of the system is not entirely
clear, but it remains repressed in both cases. The ara operon is a complex regulatory system that provides rapid and reversible responses to
changes in environmental conditions.
Genes for Amino Acid Biosynthesis Are
Regulated by Transcription Attenuation
Amino acids are required in large amounts for protein synthesis, and
E. coli has enzymes for synthesizing all of them. Not surprisingly, the
genes for the enzymes needed to synthesize a given amino acid are
generally clustered in an operon. These enzymes are needed, and
hence the operon corresponding to an amino acid is expressed, whenever existing supplies of the amino acid are inadequate for cellular
requirements. When the amino acid is in abundant supply, the biosynthetic enzvmes are no longer needed and the oneron is renressed.
Chapter 27 Regulation of Gene Expression
Trp
Trp
repressor
959
Figure 27-21 The trp operon and tryptophan biosynthesis. This operon is regulated by two mechanisms. When tryptophan levels are high (1) the repressor (upper left) binds its operator and (2) the
transcription of trp mRNA is attenuated, as described in Fig. 27-23.
Repressor
mRNA
Attenuator
trpE
trpD
trpC
-Regulatory region-
Structural genes
trp mRNA
(low tryptophan levels)
Attenuated
mRNA
(high tryptophan levels)
I
Anthranilate
synthase,
component I
Anthranilate
synthase,
[(Col)2 (CoII)2l
Chorismate
I Anthranilate
I synthase,
component II
JV-( 5' -Phosphoribosyl )anthranilate isomerase
Indole-3-glycerol
phosphate synthase
>N-( 5 '-Phosphoribosyl)- - >Enol- 1-o-carboxyanthranilate
phenylaminoPRPP PPj
1-deoxyribulose
phosphate
• Anthranilate
Glutamine Glutamate
Pyruvate
A well-defined example is the E. coli tryptophan (trp) operon,
which includes five genes for the enzymes required to convert chorismate into tryptophan (Fig. 27-21). The mRNA from the trp operon has
a half-life of only about 3 min, allowing the cell to respond rapidly to
changing needs for this amino acid. The Trp repressor is a homodimer,
with each subunit containing 107 amino acid residues (Fig. 27-22).
When tryptophan is abundant, it binds to the Trp repressor, causing a
conformational change that permits the repressor to bind its operator.
The trp operator site overlaps the promoter, and binding of the repressor blocks binding of RNA polymerase.
Here, as elsewhere, this simple "on/off" circuit mediated by a repressor is not the entire regulatory story. This system responds to different tryptophan concentrations by varying the rate of synthesis of
the biosynthetic enzymes over a 700-fold range. Once repression is
lifted and transcription begins, the rate of transcription is fine-tuned
by a second regulatory process called transcription attenuation.
Transcription attenuation describes a process in which transcription is initiated normally but is abruptly halted before the operon
genes are transcribed. The frequency with which transcription is attenuated depends on the available concentration of tryptophan. The
basis for the mechanism, as worked out by Charles Yanofsky, is the
very close coupling between transcription and translation in bacteria.
I Tryptophan
synthase,
/3 subunit
Tryptophan
synthase,
a subunit
Tryptophan synthase
• Indole-3-glycerol
phosphate
Glyceraldehyde-3phosphate
L-Tryptophan
L-Serine
Figure 27-22 Structure of the Trp repressor. The
dimeric protein is shown with the helix-turn-helix
DNA-binding motifs in red and bound molecules of
tryptophan in blue.
Chapter 27 Regulation of Gene Expression
977
activators Spl and CTF1 may act through additional bridging proteins
called coactivators. The complexity of these interactions, the number of
proteins involved, and the central role of these regulatory processes in
the life of every eukaryote ensure that this will continue to be an area
of vigorous inquiry.
Development Is Controlled by a Cascade of
Regulatory Proteins
The transitions in morphology and protein composition observed in the
development of a zygote into a multicellular animal or plant with many
distinctly different tissues and cell types involve tightly coordinated
changes in the expression of the organism's genome. More genes are
expressed during early development than in any other part of the life
cycle. For example, there are about 18,500 different mRNAs in the sea
urchin oocyte, but only about 6,000 different mRNAs in the cells of
typical differentiated tissues. The mRNAs present in the oocyte give
rise to a cascade of events that not only regulate the expression of
many genes but also determine where and when the gene products will
appear in the developing organism.
Several organisms have emerged as important model systems for
the study of development. These include yeasts, nematodes, fruit flies,
sea urchins, frogs, chickens, and mice. Our discussion will focus on the
development of fruit flies. The emerging picture of the molecular
events that occur in development is particularly well advanced in fruit
flies and can be used to illustrate patterns and principles of general
significance.
The fruit fly, Drosophila melanogaster, has a complex life cycle
that includes complete metamorphosis in its progression from an embryo to an adult (Fig. 27-35). Among the most important characteris- Figure 27-35 The life cycle of the fruitflyDrosotics of the embryo are its polarity (the anterior and posterior, dorsal phila melanogaster. In complete metamorphosis,
and ventral parts of the animal are readily distinguished) and its me- the adult insect is radically different in form from
its immature stages; this process requires extensive
"remodeling" during development. By the late embryonic stage, segments have formed from which
the various structures in the adult fly will develop.
Late embryo—segmented
Early embryo—
no segments
n t s ^g&-~.
embryonic
development
larval stages,
separated by molts
three
tar"
Larva
Day 0 Egg
Day 5
•t
fertilizatioi
Oocyte ^
pupation
Head
Thorax
/
Abdomen
metamorphosis
1 mm
(a)
(C)
Figure 27-38 Distribution of the fushi tarazu (ftz)
gene product in early embryos. In the normal embryo, the gene product can be detected in seven
bands around the circumference of the embryo, as
shown schematically in (a). These bands are seen
as dark spots (generated by a radioactive label) in
a cross-sectional autoradiograph (b), and give rise
to the segments shown here in red in the late embryo (c).
Figure 27-39 The effects of mutations in homeotic
genes, (a) Normal Drosophila head, (b) Drosophila
homeotic mutant (Antennapaedia) in which antennae are replaced by legs, (c) Normal Drosophila
body structure, (d) Homeotic mutant (Bithorax) in
which a segment has developed incorrectly to produce an extra set of wings.
homeotic genes. One well-characterized segmentation gene is fushi
tarazu (ftz), which belongs to the "pair-rule" subclass. When this gene
is lost, the embryo develops seven double-wide segments instead of the
normal 14. The mRNAs and proteins derived from the normal ftz gene
accumulate in a striking pattern of seven stripes that encircle the posterior two-thirds of the embryo (Fig. 27-38). The stripes correspond to
the positions of segments that develop later, and which are eliminated
if ftz function is lost. The expression of pattern-regulating genes such
as ftz (and bed, expressed earlier in development) establishes a kind of
chemical blueprint for the body plan that precedes the actual formation of a body structure.
Homeotic Genes Loss of homeotic genes by mutation or deletion
causes the appearance of a normal appendage or body structure at an
inappropriate body position. An important example is the
ultrabithorax (Ubx) gene. When Ubx function is lost, the first abdominal segment develops incorrectly, having the structure of the third thoracic segment. Other known homeotic mutations cause the formation
of an extra set of wings, or two legs at the position in the head where
the antennae are normally found (Fig. 27-39).
The homeotic genes span long regions of DNA. The Ubx gene, for
example, is 77,000 base pairs in length and contains introns that are as
long as 50,000 base pairs. Transcription of this gene takes nearly one
hour. The delay this imposes on Ubx gene expression is believed to be a
timing mechanism involved in the temporal regulation of subsequent
steps in development.
The precise nature of many of the events directed by these proteins, and in many cases the biochemical function of the proteins themselves, are unknown. A likely DNA-binding domain has been identified
(c)
(d)
Chapter 27 Regulation of Gene Expression
981
in a number of these proteins, however, which suggests that they are
regulatory proteins. This domain contains 60 amino acids and is called
the homeodomain because it was found first in homeotic genes. The
DNA sequence encoding this domain is called the homeobox. It is
highly conserved and has been identified in proteins from a wide variety of organisms. The DNA-binding segment of the domain is related to
the helix-turn-helix motif.
The identification of structural determinants with identifiable
molecular functions is the first step in understanding the molecular
events underlying development. As more genes and their protein products are discovered, the biochemical side of this vast puzzle will slowly
come together.
Summary
The expression of genes is regulated by a number
of processes that affect the rates at which gene
products are synthesized and degraded. Much of
this regulation occurs at the level of the initiation
of transcription and is mediated by regulatory proteins that either repress or activate transcription
from specific promoters. Regulation by repressors
and activators is called negative and positive regulation, respectively.
In prokaryotes, genes with interdependent
functions are often clustered as a single transcriptional unit called an operon. The transcription of
operon genes is generally blocked by the binding of
a specific repressor protein at a DNA site called an
operator. Dissociation of the repressor from the
operator is mediated by a specific small molecule,
called an inducer. These principles were first elucidated in studies of the lactose (lac) operon. The Lac
repressor dissociates from the lac operator when
the repressor binds to the biological inducer, allolactose.
Regulatory proteins are DNA-binding proteins
that recognize specific sequences in the DNA. Most
of these proteins have distinct DNA-binding domains. Within these domains, common structural
motifs involved in DNA binding are the helix-turnhelix and zinc finger motifs. Regulatory proteins
also contain domains for protein-protein interactions, including leucine zipper and helix-loop-helix
motifs involved in dimerization and several classes
of domains involved in the activation of transcription.
The lactose operon of E. coli also exhibits positive regulation by the catabolite gene activator protein (CAP). When cAMP concentrations are high
(glucose concentrations are low), CAP binds to a
specific site on the DNA, stimulating transcription
of the lac operon and production of lactose-metabolizing enzymes. The presence of glucose depresses
cAMP concentrations, restricting expression of lac
(and other) genes and suppressing the use of secondary sugars. Several operons that are coordinately regulated, as with CAP and cAMP, are referred to as a regulon.
Other mechanisms of regulation are also observed in prokaryotes. In the arabinose (ara) operon, the AraC protein acts as both activator and
repressor. Some repressors, as in the ara operon
and the bacteriophage A system, regulate their
own synthesis (autoregulation). Some regulatory
proteins in the ara system bind sites many base
pairs distant from each other and interact by DNA
looping mechanisms. Amino acid biosynthetic operons have a regulatory circuit called attenuation
that uses a transcription termination site (the attenuator), modulating its formation in the mRNA
by a mechanism that couples transcription and
translation and responds to small changes in
amino acid concentration. In the SOS system, multiple unlinked genes are repressed by a single type
of repressor protein, and all of the genes are induced simultaneously when DNA damage triggers
RecA protein-mediated proteolysis of the repressor. The bacteriophage A has a complex regulatory
circuit that oversees the choice between lysis and
lysogeny. Two A proteins, N and Q, act as antiterminators, modifying the host RNA polymerase so
that it can bypass transcription termination sites.
Finally, some prokaryotic genes are regulated by
genetic recombination processes that physically
move promoters relative to the genes being regulated. These diverse mechanisms permit very sensitive cellular responses to changes in environmental conditions.
Some regulation also occurs at the level of
translation. The synthesis of ribosomal proteins in
bacteria is mediated by a strategy in which one
protein in each ribosomal protein operon acts as a
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Part IV Information Pathways
translational repressor. The mRNA is bound by the
repressor and translation is blocked only when the
ribosomal protein is present in excess relative to
available rRNA.
Eukaryotes employ many of the same regulatory schemes, although positive regulation appears
to be more common and transcription is also accompanied by large changes in chromatin structure. Eukaryotic transcriptional activator proteins
are generally required for RNA polymerase binding and activity. Some transcription factors have
general functions; the TFII factors associated with
RNA polymerase II, for example, are required at
almost all RNA polymerase II promoters. Other
transcriptional activators, unique to one gene or
set of genes, have distinct domains for DNA binding and activation, and their DNA binding sites are
often found hundreds of base pairs from the site
where RNA synthesis begins.
Perhaps the most complex regulatory problem
is the development of a multicellular animal. Here,
sets of regulating genes operate in temporal and
spatial succession, turning a given area of an egg
cell into a predictable structure in the adult animal. Research continues into the molecular basis
for this highly coordinated process.
Further Reading
General
Jacob, F. & Monod, J. (1961) Genetic regulatory
mechanisms in the synthesis of proteins. J. Mol.
Ingraham, J.L., Magasanik, B., Low, K.B.,
Biol. 3, 318-356.
Schaechter, M., & Umbarger, H.E. (eds) (1987)
The operon model and the concept of messenger
Escherichia coli and Salmonella typhimurium,
RNA
were proposed in this historic paper.
Cellular and Molecular Biology, Vol. 2, American
Society for Microbiology, Washington, DC.
Nomura, M., Gourse, R., & Baughman, G. (1984)
An excellent reference source for reviews of many
Regulation of the synthesis of ribosomes and ribobacterial operons.
somal components. Annu. Rev. Biochem. 53, 75117.
Pabo, CO. & Sauer, R.T. (1992) Transcription factors: structural factors and principles of DNA recPtashne, M., Johnson, A.D., & Pabo, CO. (1982)
ognition. Annu. Rev. Biochem. 61, 1053-1095.
A genetic switch in a bacterial virus. Sci. Am. 247
(November), 128-140.
Schleif, R. (1986) Genetics and Molecular Biology,
Addison-Wesley Publishing Co., Inc., Reading, MA.
Stephens, J.C., Artz, S.W., & Ames, B.N. (1975)
Chapters 12, 13, and 14 provide an excellent acGuanosine
5'-diphosphate
3'-diphosphate
count of the experimental basis of major concepts of
(ppGpp): positive effector for histidine operon trangene regulation in prokaryotes.
scription and general signal for amino acid deficiency. Proc. Natl. Acad. Sci. USA 72, 4389-4393.
Schleif, R. (1992) DNA looping. Annu. Rev. Biochem. 61, 199-223.
Yanofsky, C. (1981) Attenuation in the control of
expression of bacterial operons. Nature 289, 751Struhl, K. (1989) Helix-turn-helix, zinc-finger, and
758.
leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends Biochem. Sci.
Zieg, J., Silverman, M., Hilmen, M., & Simon, M.
14, 137-140.
(1977) Recombinational switch for gene expression.
Science 196, 170-172.
Watson, J.D., Hopkins, N.H., Roberts, J.W.,
Steitz, J.A., & Weiner, A.M. (1987) Molecular BiRegulation of Gene Expression in Eukaryotes
ology of the Gene, 4th edn, The Benjamin/
Beardsley, T. (1991) Smart genes. Sci. Am. 265
Cummings Publishing Company, Menlo Park, CA.
(August), 86-95.
A good overview of gene regulation during developRegulation of Gene Expression in
ment.
Prokaryotes
Gottesman, S. (1984) Bacterial regulation: global
regulatory networks. Annu. Rev. Genet. 18, 415441.
DeRobertis, E.M., Oliver, G., & Wright, C.V.E.
(1990) Homeobox genes and the vertebrate body
plan. Sci. Am. 263 (July), 46-52.
Chapter 27 Regulation of Gene Expression
983
Guarente, L. (1988) UASs and enhancers: common
mechanism of transcriptional activation in yeast
and mammals. Cell 52, 303-305.
Pugh, B.F. & Tjian, R. (1992) Diverse transcriptional functions of the multisubunit eukaryotic
TFIID complex. J. Biol. Chem. 267, 679-682.
Kornberg, R.D. & Lorch, Y. (1991) Irresistible
force meets immovable object: transcription and
the nucleosome. Cell 67, 833-836.
Struhl, K. (1987) Promoters, activator proteins,
and the mechanism of transcriptional initiation in
yeast. Cell 49, 295-297.
McKnight, S.L. (1991) Molecular zippers in gene
regulation. Sci. Am. 264 (April), 54-64.
A good description of leucine zippers.
Thummel, C.S. (1992) Mechanisms of transcriptional timing in Drosophila. Science 255, 39-40.
Melton, D.A. (1991) Pattern formation during animal development. Science 252, 234-241.
Zlatanova, J. (1990) Histone HI and the regulation
of transcription of eukaryotic genes. Trends Biochem. Sci. 15, 273-276.
Ptashne, M. (1989) How gene activators work. Sci.
Am. 260 (January), 40-47.
Problems
1. Negative Regulation In the lac operon, describe
the probable effect on gene expression of:
(a) Mutations in the lac operator
(b) Mutations in the lad gene
(c) Mutations in the promoter
2. Effect ofmRNA and Protein Stability on Regulation An E. coli cell is growing in a solution with
glucose as the sole carbon source. Tryptophan is
suddenly added. The cells continue to grow, and
divide every 30 min. Describe (qualitatively) how
the amount of tryptophan synthase activity in the
cell changes if:
(a) The trp mRNA is stable (degraded slowly
over many hours).
(b) The trp mRNA is degraded rapidly, but tryptophan synthase is stable.
(c) The trp mRNA and tryptophan synthase are
both degraded rapidly.
3. Functional Domains in Regulatory Proteins A
biochemist replaces the DNA-binding domain of
the yeast GAL4 protein with the DNA-binding
domain from the A repressor (CI) and finds that the
engineered protein no longer functions as a transcriptional activator (it no longer regulates transcription of the gal operon in yeast). What might be
done to the GAL4 DNA-binding site to make the
engineered protein functional in activating gal
operon transcription?
4. Bacteriophage A Bacteria that become lysogenic for bacteriophage A are immune to subsequent A lytic infections. Why?
5. Regulation by Means of Recombination In the
phase variation system of Salmonella, what would
happen to the cell if the Hin recombinase became
more active and promoted recombination (the
switch) several times in each cell generation?
6. Transcription Attenuation In the leader region
of the trp mRNA, what would be the effect of:
(a) Increasing the distance (number of bases)
between the leader peptide gene and sequence 2?
(b) Increasing the distance between sequences 2
and 3?
(c) Removing sequence 4?
7. Specific DNA Binding by Regulatory Proteins A
typical prokaryotic repressor protein discriminates
between its specific DNA-binding site (operator)
and nonspecific DNA by a factor of 105 to 106. About
ten molecules of the repressor per cell are sufficient to ensure a high level of repression. Assume
that a very similar repressor existed in a human
cell and had a similar specificity for its binding
site. How many copies of the repressor would be
required per cell to elicit a level of repression similar to that seen in the prokaryotic cell? (Hint: The
E. coli genome contains about 4.7 million base
pairs and the human genome contains about 2.4
billion base pairs.)
8. Positive Regulation A new RNA polymerase
activity is discovered in crude extracts of cells derived from an exotic fungus. The RNA polymerase
initiates transcription only from a single, highly
specialized promoter. As the polymerase is purified, its activity is observed to decline. The purified
enzyme is completely inactive unless crude extract
is added to the reaction mixture. Suggest an explanation for these observations.